Immunostimulatory and metastasis inhibiting fermented vegetal material

ABSTRACT

The invention concerns an immunostimulatory and metastasis inhibiting fermented, dried vegetal material, pharmaceutical compositions containing the latter, the production process and the use of the dried material in the production dietary supplement and of immunostimulatory and metastasis inhibiting pharmaceutical compositions. The material according to the invention can be obtained by fermentation of wheat germ in an aqueous medium in the presence of Sacharomyces cerevisiae and by drying the fermented liquid.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/HU98/00077 which has an Internationalfiling date of Aug. 11, 1998, which designated the United States ofAmerica.

The invention relates to an immunostimulatory and metastasis inhibitingfermented and dried vegetal material. pharmaceutical compositionscontaining thereof, to the process for the preparation of the materialand the use of the dried vegetal material as a dietary supplement and inthe production of immunostimulaton, and especially metastasis inhibitingpharmaceutical compositions.

One of the main objectives of the treatment of tumours is the inhibitionof metastases. The primary tumour caused by the malignant growth of thecells spreads via metastases to the neighbouring cells and organs andcauses in the latter secondary tumours, which cannot be removedsurgically and may also become resistant to chemotherapy.

Researchers concentrate more and more on the development ofimmunomodulatory materials and materials of natural—mainly ofvegetable—origin have been studied intensively.

It is a well known fact that compounds with a quinone structure play animportant biological role. Several quinone derivatives are to be foundin plants, like ubiquinones, plastoquinones, menaquinones, which play arole in photosynthesis, but also in the cell respiratory system ofvertebra, thus also humans and in the coagulation of blood etc. Severalbenzoquinone derivatives are also used for medicinal purposes.Adriamycin, daunorubicin and mitomycin C. are quinone derivatives with acytostatic effect. While other benzo- and hydroquinones have anantimicrobal effect and are the active components of antibioticssuitable for treatments of bacterial infections like Tetran-B.Metacyclin and Doxycyclin.

The Literature reports that 2.6-dimethoxy-p-benzoquinone (2.6-DMBQ) and2-methoxy-p-benzoquinone (2-MBQ) have a fungicidal and bacteriostaticeffect, and that 2.6-DMBQ and 2-MBQ are also cytotoxic for malignanttumour cells. [Int. J. Quant. chem.: Quant. Biol. Symp. 7. 217-222(1980), 9. 27-30 (1982) and 12. 257-261 (1985); Phytochemistry 27. 225(1971) and J. Agric. Food Chem. 39. 266 (1991)]. It has been shown thata mixture of 2.6-dimethoxy-p-benzoquinone and ascorbic acid inhibits thegroh of Ehrlich ascites tumour cells in mice. [Proc. Natl. Acad. Sci.USA 81. 2088-2091 (1984) and 82. 1439-1442 (1985)].2.6-dimethoxy-p-benzoquinone and 2-methoxy-p-benzoquinone have beenfound and isolated in several plants [Magy. Kem. Folyoirat 102/7.320-325 (1996)]. These two compounds can be found in the largestquantities in glucoside form in wheat (Tricitum vulgaris), to be moreexact in wheat germ. The compounds were isolated in the fifties fromwheat germs fermented with yeast and identified for the purpose ofexamination of quality deterioration of bread to which wheat germ hasbeen added. [Hevl. Him. Acta 33., 433 (1950); J. Chem. Soc. London 1952,4821-4823].

According to the literature in wheat germ the quantity of 2-MBQ is about0.05 weight %. that of 2.6-DMBQ about 0.01 weight % (as glucoside). Thepresence of quinones as glucosides is explained by the fact thatquinones, especially) methoxy-substituted p-benzoquinones are reactivewhile their hydroquinone-glucosides are more stable and inert.

During our experiments relating to the stud, of the fermentation ofwheat germ we concluded that the dry extract deriving from the fermentedliquid produced while fermenting wheat germ with baker's yeast(Saccharomices cerevisiae) has a surprising immunostimulatory andmetastasis inhibiting effect, and it can be applied successfully as theactive agent of immunostimulatory and metastasis inhibitingpharmaceutical compositions.

This finding is surprising, because although the literature reports[Proc. Natl. Acad. Sci. USA 80. 129 (1983)] that a mixture of 2.6-DMBQand ascorbic acid inhibits the growth of Ehrlich ascites tumour cells(these are eminently suitable for quantitative analysis of the growth oftumours and for the study of their biochemical functions) the extractdescribed in the invention (which contains besides unidentifiablecomponents 2.6-DMBQ and 2-MBQ) proved to be practically ineffective onthese primary and generally all tumour cells, even when combined withascorbic acid. It proved only very effective in the treatment ofmetastases. The aspects of the treatment of primary tumours and themetastases developing from the former are basically different, so it wasnot self-evident for the researcher that a plant extract containingcomponents which are known to be effective in the treatment of primarytumours and not yet fully identifiable as to its chemical compositionshould have an immunostimulatory and metastasis inhibiting effect.[Cancer. Principles and Practice of Oncology, Vol 1.4^(th) edition. J.B. Lippincott Company. Philadelphia, 1993.]

Thus the present invention concerns an immunostimulatory and metastasisinhibiting fermented and dried vegetal material which is obtainable byfermenting wheat germ with baker's yeast in an aqueous medium filteringthe fermented liquid cell-free and drying it.

The present invention also relates to an immunostimulatory andmetastasis inhibiting pharmaceutical composition the active agent ofwhich contains the fermented, dried vegetal material described in theinvention.

The present invention also concerns the use of the above mentionedfermented and dried vegetal material in the production ofimmunostimulatory and metastasis inhibiting pharmaceutical compositions.

The present invention also concerns the processes of treating mammalsfor the stimulation of the immune system and inhibition of metastaseswith the pharmaceutical composition described in the invention.

The invention further concerns a dietary supplement comprising thefermented, dried vegetal material described in the invention andmaltodextrin.

According to the invention the fermented, dried societal material hasbeen identified and examined on the basis of its 2.6-DMBQ content. Wewould like to note here that full identification of the chemicalcomposition of the extract was not possible with the available methods,so all data concern the 2.6-DMBQ contents as a basis.

It proved suitable to use for the production of the fermented, driedvegetal material described in the present invention wheat germ—which isa by-product of flour milling and available in large quantities—aroundto flour quality, either in its original state or defatted. The use ofdefatted wheat germ has no specific advantages. Fermentation was donewith baker's yeast (Saccharomyces cerevisiae). This type of yeast iscommercially available. The period of fermentation seas about 10-24hours, preferably 15-20 hours or about 18 hours. The temperature of thefermentation is about 25-35° C. preferably about 30° C. The weight ratioof the wheat germ and the yeast is 4:1-2:1, preferably about 3:1, theweight ratio of the dry matter and water is 1:6-1:12, preferably about1:9.

The fermentation—on a laboratory scale—can be e.g. effected by adding ina glass fermentor to the freshly ground wheat germ the yeast suspensionin water, and by stirring or shaking the mixture. The fermented liquidgets foamy.

After fermentation the mixture is centrifuged for 5-15 minutes at2000-4500/min, preferably about 3000/min. The supernatant is boiled,cooled and dried in an appropriate manner, e. g. by lyophilisation orspray-drying.

The product, a reddish-brown powder is the material according to theinvention. It is expedient to keep the material until further usecooled, and because of its hygroscopic character in sealed containers.The 2.6-DMBQ contents of the resulting dry material is about 0.4 mg/g.

In a large scale fermentation (e.g. 4 cubic meters) it is suitable toapply continuous areating, e.g. 0.5 l air/l of fermented liquid/minute,and slow stirring. The period of fermentation is about 18 hours. Inorder to inhibit foaming the usual additives can be applied—it ispreferable to use sunflower oil. At the end of the fermentation processareating and stirring are discontinued and the fermented liquid isseparated from the wheat germ—yeast suspension in the usual manner, forexample in a screw decanter, subsequently in a separator and a filterpress. If necessary—a filter aid material can be added. It is suitableto use 5-10 kg filtering perlite per cubic meter of fermented liquid.The fermented liquid is filtered sharp, and the quality of the filteringis checked by a microscope. The filtered fermented liquid containspractically no cells, which means that no more than 1 yeast cell isfound per 10 sights. The resulting fermented liquid, Which containsabout 1.5 % by weight dry material is evaporated in a vacuum condenser,preferably at a temperature of about 40-50° C. and after terminating thevacuum is boiled at atmospheric pressure for about minutes. This resultsin a decrease of harmful enzyme activities. Subsequently the mixture isdried by spray drying, e.g. in a rotating spray apparatus. If the abovefermentation process is used the 2.6 DMBQ content of the resultingfermented, dried vegetal material is 0.12-0.52 mg/g dry material and the2 MBQ content is 0.05-0.28 mg/g dry material—depending on thebenzoquinone content of the wheat germ used.

As the final product is hygroscopic in order to enhance theeffectiveness of the spray drying and the use of the final product oneof the usual additives, e.g. maltodextrin, acacia gum, guar gum,xanthan, locust bean flour etc. can be used during spray drying. It ispreferable to use maltodextrin. The suitable process is to determine thedry material content of the mixture evaporated in the vacuum condenserand boiled and add so much maltodextrin that the dry material content ofthe mixture to be dried is about 30% by weight. It is suitable todissolve the maltodextrin in hot water and add it cooled to thecondensed mixture. After drying the final product—a powder—containsabout 60% by weight fermented, dried vegetal material and about 40% byweight maltodextrin.

The stability of the obtained material can be checked by monitoring thechanges of the 2.6 DMBQ concentration. Quantitative analysis can be doneby HPLC. The powder produced according to the above described processremains stable for about 3 years at room temperature. The fermented,dried vegetal material according to the invention can be used as theactive agent of immunostimulatory and metastasis inhibitingpharmaceutical compositions. The pharmaceutical composition may alsocontain ascorbic acid or other cytostatic materials beside the saidactive agent.

The fermented, dried vegetal material can be processed to the usualsolid or liquid pharmaceutical compositions for peroral or parenteraladministration. During the production of the pharmaceutical compositionsthe hygroscopic character of the fermented, dried vegetal material mustbe taken into account. A suitable form is a capsule which protects theactive agent from the humidity of the air.

In the production of the pharmaceutical compositions auxiliary materialsusually applied in pharmaceutical practice can be used. As thesematerials and their possible uses are described in the pharmaceuticalliterature in detail, the selection and preparation of the suitable formis a routine task. The one time dosage of the effective agent can varybetween wide limits depending on the state of the patient, and theselection of the suitable effective dosage must always be theresponsibility of the doctor. Generally suitable results can be obtainedif the dosage is 0.001-100 g, preferably 0.01-50 g, even more preferably0.1-40 g per kg of body weight, e.g. in dosages of 0.1-10, 1-25 or 10-30g.

The fermented, dried vegetal material according to the invention canalso be mixed with ascorbic acid (Vitamin C). According to ourexperiments ascorbic acid may enhance the metastasis inhibiting effectof the material according to the invention. The weight ratio of thefermented, dried vegetal material and ascorbic acid can be e.g.10:1-1:1, preferably 6:1-2:1, more preferably 3:1, 4:1 or 5:1.

The invention also concerns the immunostimulatory and/or metastasisinhibiting treatment with the fermented, dried vegetal materialaccording to the invention. The point of the treatment is to give thepatient an effective dose of the fermented, dried vegetal material.

The fermented, dried vegetal material can also be used as a dietarysupplement for mammals. In this case it is preferable to apply themixture containing maltodextrin and the usual auxiliary materialsapplied in food industry, e.g. aromatic materials, sweeteners, colouringagents etc. The dietary supplement can e.g. be produced by granulatingthe mixture containing 60% by weight dried matter and 40% by weightmaltodextrin plus aromatic materials and sweeteners in a fluid bed, andpacking the instant granulate in e.g. bags.

The attached figures are described below:

FIG. 1. Calibration diagram of the HPLC measurement of 2.6 DMBQ

FIG. 2. HPLC chromatogram of the chloroform extract of the material

FIG. 3. HPLC chromatogram of the ethanol extract of the material

FIG. 4. Binding of B16 tumour cells in the presence of lyophilisate ofdifferent concentrations in the 60th and 90th minute after placing.(Every value represents the average of 8 parallel measurements±SD,evaluation by spectrophotometric measurement)

FIG. 5. The effect of different dosages of lyophilisate on theproliferation of B16 cells after 24 and 48 hours after the commencementof the treatment. (Every value represents the average of 8 parallelmeasurements±SD, evaluation by spectrophotometric measurement)

FIG. 6. The development of A2058 human melanoma in the presence ofdifferent dosages of lyophilisate 24 hours after the commencement of thetreatment. The evaluation of the cultures was performed on the basis ofprotein (SRB) and dehydrogenase activity (MTT) by parallelspectrophotometric measurements. (Even value represents the average of 8parallel measurements±SD)

FIG. 7. Ration of apoptotic cells (FACS analysis) in the A2058 humanmelanoma in vitro cultures after treatment With different dosages of thelyophilisate.

The following examples serve to illustrate the fermented, dried vegetalmaterial according to the invention, its production and pharmacologicaleffects.

EXAMPLE 1 Laboratory Scale Fermentation of Wheat Germ

A suspension of 33.3 g yeast (Saccharomyces cerevisiae) and 1000 ml ofdrinking stealer have been added to 100 g fresh wheat germ (according tothe Hungarian standard MSZ-081361-80) ground to flour quality. Themixture was shaken in a shaker for 18 hours at 30° C. During this periodthe fermented liquid got foamy and reached about three times itsoriginal volume. After fermentation the mixture was centrifuged for 15minutes at 3000/min. After boiling and cooling the supernatant was driedby lyophilisation, and the resulting lyophilized matter was kept untilfurther use in the freezer (−10° C.). The 2.6 DMBQ content of theresulting lyophilisate was 0.4 mg/g dry material (0.04% by weight).

EXAMPLE 2 Large Scale Fermentation of Wheat Germ

300 kg, wheat germ ground to flour quality (according to the Hungarianstandard) and 100 kg, yeast were placed in a 5 cubic m fermentor, anddrinking water was added until the volume became 4000 1. Thefermentation period was 18 hours, during which continuous areating(0.5.1 air/l fermented liquid/minute) and slow stirring (30 rev./min)was used. In order to inhibit foaming 1 l/cubic m sunflower oil wasadded to the mixture. After fermentation areating and stirring werediscontinued, and the fermented liquid was separated first in a screwdecanter, then in a separator and finally in a filter press with textilefilter. As auxiliary material 10 kg filtering perlite/cubic m was added.The fermented liquid was filtered sharp and the sharpness was checked bemicroscope. The filtered fermented liquid contained practically nocells, which meant that maximum 1 yeast cell was found per 10 sights.The resulting fermented liquid, which contained about 1.5% by weight drymaterial was evaporated in a vacuum condenser, at a temperature of40-50° C. and after discontinuing the vacuum boiled at atmosphericpressure for about 15 minutes. After this the dry material content ofthe solution was determined and so much maltodextrin—first solved in hotwater and then cooled—was added that the dry matter content of thesolution became about 30 mass %. After this the solution was spray driedin a shear nozzle rotating spray drier in which the temperature of theoutgoing air was 90° C. The resulting final product—a powder—contained60% by weight of the fermented vegetal material according to theinvention and 40% by weight maltodextrin. The 2.6 DMBQ contentwas—determined by HPLC according to the method described in thefollowing example—0.4 mg/g dry material.

EXAMPLE 3 Characteristics of the Material According to the Invention

The material according to the invention can be characterised in twoways, either by determining its 2.6. DMBQ content or by the so-calledfingerprint chromatogram. In both cases HPLC chromatography is used.

Analysis has been performed both on the material produced as describedin Example 1 and on that obtained as described in Example 2, and theresults were in both cases identical.

A. Quantitative and Qualitative Analysis of Benzoquinone Derivatives

Preparation of the sample

Before analysis it became necessary to increase the concentration ofbenzoquinones in the lyophilisate. In order to achieve this thelyophilisate was diluted to the original concentration with distilledwater (1 weight % dry manner content)−(0.5 g lyophilisate, 50 mldistilled water). The solution was extracted with 3×25 ml chloroform inthree steps. Eventual humidity remaining in the chloroform phase wasremoved with water-free Na sulphate. After filtering the chloroformphase was evaporated to the rest chloroform was added to a total volumeof 5 ml. This sample was injected during the HPLC analysis.

Qualitative and quantitative analysis of the benzoquinone derivativeswas performed by the HPLC method.

Description of the HPLC method

The applied HPLC equipment consists of a Beckman model 114 M pump, aLaborMim UV respectively a Merck-Hitachi-DAD mod. 4500 diode arraydetector, and a Waters 740 type integrator unit. A Chromsil C18 (250×4mm) 10 μ column was used for measurements. UV detection was performed ata frequency of 290 mm, the flow rate was 2 ml/min.

The composition of the applied eluent was the following: Na₂HPO₄25 mmol.Na₂HPO₄25 mmol. Na₂EDTA 25 mmol. NH₂OH.HCl 20 mmol, 10 volume %methanol. pH=6.05.

Three different benzo—and hydroquinones were analyzed by the methoddescribed above. There is but a small difference between the retentiontime of the compounds. By decreasing the strength of the eluent—theorganic phase to 10%—selectivity was enhanced to a great extent.Analysis of the retention data has shown that p-benzoquinones show agreater retention than the corresponding hydroquinones and retentionincreases with the number of methoxy groups. The concentration of allthree standards was 0.1 mg/ml. Table 2, shows the retention times(t_(R)) of the standards and the capacity factor (k′) data. The aim ofthe measurements was detection and quantitative definition of2.6-dimethoxy-p-benzoquinone. So in the following this part of themeasurements will be discussed in detail. It was examined whether theelaborated method is suitable for quantitative analysis of2.6-dimethoxy-p-benzoquinone. The calibration diagram used is shown inFIG. 1. With a correlation coefficient of 0.99 the result is a straightline. It was also checked whether the measurements can be reproduced, onevery sample three parallel measurements were conducted and thescattering of the results was calculated. Table 1, shows the parallelresults of measurements conducted on samples during a period of sixmonths and their scattering. Our results show that the measuring methodis suitable for exact and reproducible measurement of2.6-dimethoxy-p-benzoquinone.

TABLE 1 Parallel values of the quantitative analysis of 2.6-DMBQ and thescattering of the results 2.6-DMBQ content Date (mg/50 g of sample)(σ-%) 04.27. 3.45 3.54 3.66 0.08-2 04.28. 3.27 3.68 3.31 0.18-5 05.10.1.56 1.49 1.48 0.03-1.9 05.10. 3.65 3.42 0.12-3 06.08. 3.00 2.98 2.860.06-2 06.09. 2.92 3.06 2.87 0.08-2.3 06.10. 3.34 3.23 3.36 0.05-1.5

TABLE 2 Retention time (t_(R)) and capacity factor (k′) values of theexamined materials Standard t_(R) k′ 2.6-DMBQ 13.4 5.0 2.6-DMHQ 5.1 1.22-MBQ 8.5 2.7

FIG. 2 shows the HPLC chromatogram of the chloroform sample prepared asdescribed above. The chromatogram shows only one peak characteristic for2.6 DMBQ. The 2.6 DMBQ contents of the sample was determined on thebasis of the laner.

B. Fingerprint Chromatogram

Preparation of the sample

50 ml 96% of volume ethanol was added 5 g of spray dried material(prepared as described in example 2.). The mixture was shaken for 30minutes at a temperature of 50° C. and 200/min. After this the mixturewas filtered, evaporated dry and the remaining material solved in 10 mlof methanol. The filtered solution was injected onto the column.

HPLC method

The HPLC instruments column and conditions described above are usedhere, too, but the composition of the eluent was the following:Na₂HPO₄1.25 mmol, NaH₂PO₄1.25 mmol. Na₂EDTA 1.25 mmol, NH₂OH.HCl 2.50mmol. 5 volume % methanol.

FIG. 3. shows the resulting HPLC chromatogram. It is shown that underthese conditions two characteristic peaks appeared, one at about 4.7minutes (5) and one at 5.8 minutes (6). At a retention time of 7.3.resp. 7.7 minutes two peaks (7.8) followed each other quickly, whichcannot be separated completely. A peak of smaller intensity appeared at9.8 minutes (9), followed by a characteristic intensive peak (11) atabout 13.1 minutes. At about 15 minutes a smaller peak (12) and anotherintensive peak (14) at 18 minutes. At 21.8 minutes the chromatogramshowed a smaller peak (15) and at around 31 minutes a flatter peak,containing several materials.

C. Stabilit Tests

The decomposition of the material according to the invention was checkedvia the chanqes in concentration of the 2.6-dimethoxy-p-benzoquinone. Weconducted storage experiments at three different temperatures (roomtemperature 20° C. 40° C. and 60° C.) The lyophilisate—about 1 g—asstored in test-tubes which were sealed air-tight. The duration of theexperiments was 8 weeks, every week three parallel measurements wereconducted on samples taken from all three series kept at differenttemperatures. The quantitative analysis of benzoquinone derivatives wasconducted by HPLC.

The tests have shown that the dried material according to the inventionremained stable even after three years at room temperature, that is, the2.6 DMBQ content remained practically unchanged. At the same time thematerial is not stable at 40° C.—the 2.6 DMBQ decomposes in a fetalweeks, and at 60° C. the 2.6 DMBQ contents decreases rapidly within afew days.

The decomposition of 2-methoxy-p-benzoquinone was also studied in bothseries of measurements. This compound is more instable than2.6-dimethoxy-p-benzoquinone, and as a consequence its presence couldnot be shown after one week of storage in the samples kept at 40 or 60°C. At room temperature the concentration of this compound remainedunchanged, too.

EXAMPLE 4 Tests on Effectiecness

Both the lyophilisate prepared as described in example 1, and the spraydried material prepared as described in example 2, have been tested foreffectiveness. A standardised dried material was used with a 2.6 DMBQcontent of 0.4 mg/g dry material and a HPLC curve as the one shown inFIG. 2. Both materials gave the same results—so the material accordingto the invention will be called further simply lyophilisate. Below theresults of the biological and toxicological tests concerning thislyophilisate will be discussed, with a special stress on the immunereconstitution and tumour growth and metastasis inhibiting effects.

1. Tumour Growth and Metastasis Inhibiting Effect (in vivo tests)

For the tests the following injectable tumour types growing in mice orrats were used: a highly metastasis forming variant (3LL-HH) of Lewislung carcinoma (mouse iung cancer), B16 mouse melanoma and HCR-25 humancolon carcinoma xenograft.

The 3LL-HH (LLT-HH) and B16 tumours were sustained in C57Bl/6 inbredmice. The HCR-25 xenopraft was injected into CBA/CA mice, previouslyimmunesuppressed by thymectomy and complete body irradiation. In thecase of 3LL-HH and HCR-25 tumour the tumour cells were transplanted intothe spleen, in the case of B16 melanoma into the muscles of the leftlower extremity. A treated and a control group of the animals injectedwith HCR-25 tumour were also splenectomized 21 days after thetransplantation of the tumour.

Treatment with he lyophilisate described in the invention was started 24hours after injection of the tumour. Daily dosages of 3 g/body weight kgwere administered per os. with a stomach probe, in the form of 0.6 g/mlaqueous suspension.

The tests were terminated 14 days after transplantation in the case ofthe 3LL-HH tumour, 21 days after transplantation in case of the B16tumour and 51 days after transplantation in the case of HCR-25 5 tumourby bleeding the animals to death under narcosis.

Tables 3, 4, and 5, summarise the results of the tests.

TABLE 3 Effect of treatment with the lyophilisate according to theinvention on the number of liver metastases in case of 3LL-HH mouse lungcancer injected into the spleen Treatment Number of injected cellsNumber of liver metastases Control 3 × 10³ 104.0 ± 28.2  Lyophilisate 3× 10³ 29.8 ± 16.4* p < 0.01 every test group contained 10 animals

TABLE 4 Effect of treatment with the lyophilisate according to theinvention on the number of liver metastases 51 days after the HCR-25human colon carcinoma had been injected into the spleen ofimmunesuppressed CBA/CA mice Weight of spleen Number of Treament withtumour (g) liver metastases Control, non-splenectomized 1.02 ± 0.59 42.0± 25.8 Lyophilisate, 0.62 ± 0.47 19.5 ± 19.0 non-splenectomized Control,splenectomized* 0.10 ± 0.02 19.1 ± 13.5 Lyophilisate 0.08 ± 0.02 10.6 ±11.6 splenectomized* *Splenectomy was performed 21 days after the tumourhad been injected onto the spleen Every test group contained 12 animals

TABLE 5 Effect of treatment with the lyophilisate according to theinvention on the weight of the extremity injected with the tumour and onthe number of lung metastases in the case of B16 melanoma injected intothe muscles of the extremity 21 days after the tumour had been injectedWeight of the extremity with Number of lung metastases Treatment tumour(average) (average) Control 7.6 ± 0.43 42.4 ± 10.2 Lyophilisate 7.2 ±0.38  6.2 ± 3.7* *p < 0.01 every test group contained 10 animals

The above results show that treatment with the lyophilisate described inthe invention decreased significantly—by 71%—the number of lungmetastases when 3LL-HH tumour had been injected into the spleen. (Table3.)

In case of HCR-25 human colon carcinoma the 50 day treatment decreasedboth the weight of the spleen with tumour and the number of livermetastases. The number of metastases as compared to the control groupwas about 50% both in the splenectomized and in the non-splenectomizedgroups. (Table 4.)

In case of B16 melanoma injected into the muscle the weight of thetumour growing in the muscle did not change as an effect of thetreatment, but the number of lung metastases decreased verysignificantly, by 85% as compared to the control group (Table 5.)

II. In Vitro Tests

As the above tests have shown unambiguously that the lyophilisatedescribed in the invention can decrease significantly metastases ofmalignant tumours, the effects of the products on the different phasesof the forming of metastases were also examined. The forming ofmetastases consists of several phases, in which besides theproliferation and apoptotic activity of the cells of the primary tumour,the adhesion potential of the tumour cells and the protective mechanismsof the organism aeainst the tumour cells play a role. In the in vitrotests the effect of the lyophilisate on cell proliferation and apoptosisand adhesion were studied.

As a great part of the in vivo tests was performed on B16 mouse melanomacells, in the first step this tumour was used to test the lyophilisate.

1. Adhesion Test

The adhesion of the tumour cells was tested on a 96-hole microplate. Thedosages of the lyophilisate were 300, 3000 and 30000 μg/ml. RPMIculture-medium was used, both in the absence of serum and in thepresence of 10% FCS. The adhesion was tested 10, 30, 60, 90 and 120minutes after incubation in usual circumstances. Evaluation wasperformed by colorimetry process on the basis of the SRBassay.(Mossmann. T.: 3. lmmunol. Neth. 65, 55-63/1983/). The test isbased on sulforhodamine B-colouring of the total protein content of theculture, absorbency was read at 570 nm with a spectrophotometer. FIG. 4.only shows two moments in time, which, however represent the effect ofthe lyophilisate adequately. It was shown that if the dosage of thelyophilisate was 3000 μg/ml or ten times higher, it decreased theadhesion of tumour cells dramatically, both in the presence and absenceof serum. If the dosage is 300 μg/ml, no such effect can be observed.

2. Proliferation Test

In this test the tumour cells were placed 24 hours before treatment onthe 96-hole microplate. After treatment with appropriate dosages of thelyophilisate the proliferation activity of the cells was also tested bythe SRB assays, 24, 48 and 72 hours after treatment. The results of therepeated tests have shown that as effect of the treatment in the 900-15000 μg/ml rangle the tumour cells come to the surface of the monolayerand—as shown by the trypan-blue dye exclusion method—died. (Kaltenbach.J. P. et al.: Exp. Cell Res. 15, 112-217/1985/). (FIG. 5.)

Our tests on human amelanotic melanoma (A2058 tumour cells—Todaro, G. J.et al.: Proc. Natl. Acad. Sci. USA 77, 5258-5262/1980/) have shownsimilar results to those on mouse melanotic melanoma (FIG. 6.) In thecase of this tumour parallel to the SRB assay the MTT assay representingthe metabolic activity of the cell was also performed (Cole, S. P. C.:Cancer Chemother. Pharmacol. 17, 29-263/1986/). The test is based on thephenomenon that a metabolically, active cell transforms tetrasolium saltinto a coloured formazan product, primarily via its dehydrogenaseactivity. The colour reaction, which is proportional to the activity,can be read by a spectrophotometer at 570 nm, the MTT assay has shownclearly that functional activity of the tumour cells decreased even ifthe dosage was 300 μg/ml (FIG. 4.). As in case of the cells come to thesurface the cause of apoptosis was unknown, the apoptotic activity ofthe complete cell population was tested by flow cytometric (FACS)analysis (FIG. 7.). As FIG. 7 shows the tumour cells were inapoptosis—depending on the dosage—to an unusually great extent.

III. Test of Effects on Immune Reaction

The effect of the lyophilisate described in the invention was examinedin two different models. In one series of tests the possibility of blasttransformation of mononuclear cells gained from the spleen of animalstreated with the lyophilisate was studied, while in the others in theallograft skin transplantation model the total binding of skintransplanted in the back region of mice was studied.

1. Test of Blast Transformation of T-lymphocytes

Treatment with the lyophilisate described in the invention significantlyincreases blast transplantation of the T-lymphoctes playing an importantrole in immune reaction. This was shown by the following experiment.

C₅₇Bl₁₆ mice were treated for six weeks five times per week per os. witha stomach probe with the lyophilisate described in the invention in a 3g/kg dosage (0.6 g/ml aqueous suspension). After completion of thetreatment the lymphocytes gained by perfusion from the spleen of theanimals were transferred into a cell culture, treated with 1 μg/ml ConA. After 48 hours the cells performing DNS synthesis were marked by 0.4μCi ³H timidin. The decree of marking was defined with a liquidscintillation counter. (Beckman). As Table 6, shows treatment with Con Asignificantly increased—as compared to the control—the incorporation of³H-TdR, that is, blast transformation.

TABLE 6 Incorporation of ³H-timidin into the spleen lymphocytes of thecontrol and lyophilisate treated mice 1 μg/ml ConA Treatment average(cpm) SEM Control 3760.6 583.3 Lyophilisate 8041.8 957.1

2. Test of Immunostimulator Effect in the Allograft Skin TransplantationModel

The best model for the illustration of the restitution of deficientimmune reaction is the allograft skin transplantation test in mice madepartdy immune deficient by thymectomy (operational removal of thethymus). The C₅₇B1 ₁₀ and B₁₀LP mouse stocks only differ in the H-3locus, so the skin transplanted from members of one stock onto the otheris not rejected within 7 days, but only after about 3 weeks. If therecipient had been thymectomized, rejection occurs after an average of50 days. All materials which advance the ripening and differentiating ofthe lymphocytes of the medulla, like the hormones of the thymus,decrease the time necessary for rejection of the transplanted skain. (J.Immunpharmacology 7, 67-78/1985/). The same effect could be observed inour tests when treatment with the lyophilisate described in theinvention was applied.

C₅₇Bl₁₀ mice were used as recipient, and B₁₀LP mice as donor. Therecipient mice were thymectomized and skin transplantation followedafter 7 weeks. Per os −5 times per week, 30 mg/kg—treatments with thelyophilisate were stared one week after thymectomy. Treatment wasconcluded 70 days after skin transplantation. Eventual rejection of theskin was observed daily. Table 7 shows that rejection time innon-thymectomized mice was 21 (males) resp. 28.7 (females) davs. Inthymectomized mice rejection time was lengthened to 52.4 resp. 41.6days. Treatment with the lyophilisate according to the inventionshortened significantly in thymectomized and treated mice the survivalof the skin transplants (grafts). This shows that immune deficiencycaused by thymectomy was reduced signlficantly as a result of thetreatment, which means that the lyophilisate has an immune stimulanteffect.

TABLE 7 The effect of treatment with the lyophilisate according to theinvention on the rejection of skin grafts (recipient C₅₇Bl₁₀ mice, donorB₁₀LP mice) Rejection time Male Female Treatment average (days) SEMaverage (days) SEM Control (non- 21.0 3.1 28.7 4.5 thymectomized)Control 52.4 5.0 41.6 5.5 (thymectomized) Lyophilisate 28.8* 8.6 32.6**4.5 (30 mg/kg) *0.001 < p < 0.01 vs thymectomized control **0.01 < p <0.05 vs thymectomized control

In vivo and in vitro tests with the lyophilisate according to theinvention show that this product has a significant antimetastatic effectin several animal test models. This effect is probably connected to theimmunostimulator, effect observed both in the in vivo and in vitrotests, but it is possible that the decrease in the number of metastasesis also influenced by the antiproliferative, apoptosis causing effects,the effect of the material on adhesion and the effect causing thecreation of free radicals.

IV. Test of Radical Binding Activity

As benzoquinones have got a well-known effect on the forming of freeradicals, the radical binding activity, of the lyophilisate described inthe invention was also tested. Both the superoxide (SSA) and hydroxylradical binding (OH-SA) were measured with the electron spin resonancemethod. The lyophilisate has a significant SSA, the clean radicalbindings activity of 1 mg corresponds to the activity of 5.64 μgsuperoxide dismutase (SOD). The lyophilisate has no OH-SA activity, butit disrupts the hydrogen-peroxide/Fe hydroxyl radical forming system, soit can be supposed that it has so-called non-chelator activity.

V. Toxicological Tests (Subacute)

The 77 day toxicological tests were performed according to therecommendations of the Registry of Industrial Toxicology Animal-data(RITA). (Exp. Toxic. Pathol. 47, 247-266/1995/) on F344 rats and C₅₇Bl₁₆mice. The animals were treated daily with a dosage of 3 g/kg (0.6 g/mlaqueous suspension). During treatment the changes of weight of theanimals, eventual pathological physical changes, spontaneous perishingof the animals were observed. On concluding the test the weight of theheart, lungs, thymus, spleen, liver, kidneys and testicles were measuredand the 34 organs prescribed by the RITA were pathologically examined.No spontaneous perishing was observed, the weight of the animals changedlike that of the control group. On conclusion of the test the weight ofthe different organs showed no changes as compared to the control group.During pathological processing of the treated animals no changes wereobserved, which could have been caused by the lyophilisate.

The above results show that the fermented vegetal material according tothe invention is not toxic, and because of its immunostimulatory effectit is indicated in all states in which the irmune system is damaged.Because of its above described biological characteristics it can haveits complementary uses in the medical treatment of malignant tumours,mainly in the inhibition of metastases.

What is claimed is:
 1. A process for producing an immunostimulatory andmetastasis inhibiting fermented, dried material wherein ground wheatgerm is fermented in an aqueous medium in the presence of Sacchromycescerevisiae and the fermented liquid is separated, fine filtered,evaporated, boiled and dried.
 2. The process according to claim 1,wherein the fermentation is carried out at a temperature of about 30° C.for about 18 hours under continuous areating and stirring.
 3. Theprocess according to claim 2, wherein the drying is performed in thepresence of maltodextrin.
 4. A process according to claim 1, wherein theliquid is dried in the presence of auxiliary drying materials.
 5. Amethod for the production of an immunostimulatory and metastasisinhibiting pharmaceutical composition wherein ground wheat germ isfermented in an aqueous medium in the presence of Saccharomycescerevisiae and the fermented liquid is separated, fine filtered,evaporated, boiled, dried and added to a pharmaceutically acceptablecarrier.
 6. A method for the production of a dietary supplement formammals wherein ground wheat germ is fermented in an aqueous medium inthe presence of Saccharomyces cerevisiae and the fermented liquid isseparated, fine filtered, evaporated, boiled and dried and adding thedried composition to an auxiliary material applied in the food industry.